Mechanism Of Exosomal MiR-451 Defencing Against Mycoplasma Gallisepticum(HS Strain) Infection By Targeting YWHAZ In Chickens

Chronic respiratory disease(CRD),caused by Mycoplasma galisepticum(MG),is a highly contagious respiratory disease.Chickens of different breeds,genders,and ages are infected year-round.The disease is endemic in all poultry-producing countries and causes huge economic losses to the poultry industry.As a result,seeking effective methods to prevent and control CRD has become a major scientific and practical problem to be solved urgently.Studies have shown that exosomes carry miRNAs with the circulatory system and transfer to adjacent or distant cells or tissues and play an important role in the occurrence and development of diseases.Therefore,in-depth studies of exosome-miRNAs is necessary to elucidate the pathogenesis,diagnosis and treatment of MG.In this study,the exosomes derived from chicken type ? pneumocytes(CP-?)were isolated and identified,and the differentially expressed exosome-miRNAs derived from MG-HS-infected and uninfected CP-? were screened by Hi Seq2500 sequencing.The role and molecular mechanism of gga-miR-451 in MG-HS infection were further explored.The main findings are as follows:1.Construct a model of MG-HS-infected CP-?,collect the cell supernatant,and extract the exosomes by ultracentrifugation.The exosomes were isolated from the media and collected from MG-HS-infected and non-infected CP-? by high-speed centrifugation and were characterized by NTA and TEM.These results showed an average particle diameter of 30–150 nm,double-layered membrane structure of round-shaped vesicles.The exosomal surface markers CD9 and CD63 could be detected using Western blot,and was expressed significantly higher in the MG-HS infection group than the control group.It shows that MG-HS infection promote secretion of exosomes derived from CP-?.2.Among these differentially expressed miRNAs(DEGs),only 30 miRNAs(9 up-regulated and 21 down-regulated)were significantly differentially expressed(fold-change >1)in the MG-HS-infected vs.non-infected groups.A target prediction of DEGs was performed using miRanda,Pita,and RNAhybrid.After performing GO and KEGG functional annotation of target genes,we found that the predicted targets of DEGs are mainly involved in regulating cell cycle,apoptosis,MAPK,and Toll-like receptor signaling pathways.To validate the reliability of the sequencing results,RT-q PCR results showed that the expression of gga-let-7d,gga-miR-451,gga-miR-133-3p and gga-miR-223 were significantly downregulated,gga-miR-193a-3p and gga-miR-33-5p were up-regulated,and gga-miR-460b-5p and gga-miR-202-5p had no significant differences between the MG-HS-infected group and control groups.The sequencing coverage of the exosome-miRNAs was 75%,and the small RNA-seq results were valid.3.RT-q PCR showed that gga-miR-451 was significantly up-regulated in chicken embryo lung tissue at 6d,10 d,and 11 d after MG-HS infection in CP-II and DF-1 cells while gga-miR-451 level was significantly decreased in the exosomes derived from MG-HS-infected CP-?(3.8-fold),indicating that gga-miR-451 was selectively loaded into exosomes derived from MG-HS-infected CP-?(Exo-MG).CP-?-derived exosomes were labeled with PKH67.They were then incubated with DF-1 cells for 6 h,and observed by confocal microscopy,which revealed the incorporation of exosomes into DF-1 cells.At 36 h post-incubation,RT-q PCR showed that gga-miR-451 was significantly decreased in Exo-MG as compared to the control group.Those suggest that the selective secretion of gga-miR-451 plays an important role in MG-HS infection.4.To reveal whether gga-miR-451-absent exosomes derived from MG-HS-infected CP-? are involved in the regulation of inflammation in DF-1 cells,TNF-? and IL-1? protein levels were detected by ELISA at 36 h post-incubation.Exo-MG could increase TNF-? and IL-1? protein levels in DF-1 cells significantly,indicating that Exo-MG promotes its inflammatory response against MG-HS infection after entering the recipient cells.However,the inhibition of endogenous gga-miR-451 significantly increased the protein levels of TNF-? and IL-1? and IL-6,the overexpression of gga-miR-451 significantly down-regulated the protein levels of TNF-?,IL-1?,and IL6 in MG-HS-infected DF-1 cells.Those show that the inhibition of gga-miR-451 promotes the inflammatory response of normal cells,while the overexpression of gga-miR-451 inhibits the inflammatory response of infected cells.5.We used publicly available prediction software/servers and performed dual-luciferase reporter assay,the results showed that YWHAZ is a candidate target gene for gga-miR-451 and the overexpression of gga-miR-451 significantly down-regulated the dual-luciferase activity of wild-type vectors but not the luciferase activity of the mutant vectors.RT-q PCR and WB further revealed that the overexpression of gga-miR-451,but not miR-451-NC,resulted in a significant reduction in YWHAZ at both the m RNA and protein levels,and the m RNA and protein levels of YWHAZ were both significantly down-regulated in MG-HS-infected DF-1 cells and chicken embryo lung tissues at 6d,10 d,and 11 d after infection.These data demonstrate that YWHAZ is a functional target gene of gga-miR-451.Further analysis by RT-q PCR and WB showed that gga-miR-451-absent exosomes derived from MG-HS-infected CP-? significantly increased the protein levels of YWHAZ but not m RNA levels as compared to the control group.This indicates that both exosomes and intracellular gga-miR-451 negatively regulate YWHAZ in cells.6.The results of CCK-8,cell cycle kit,and apoptosis kit showed that gga-miR-451 inhibited the proliferation of MG-HS-infected DF-1 cells,generated cell cycle changes with a larger proportion of cells in the G1 phase and a smaller proportion of cells in the S and G2 phases compared with miR-451-NC,and promoted cell apoptosis.The results of RT-q PCR and WB showed that the overexpression of gga-miR-451 significantly reduced p MGA1.2 expression at the m RNA and protein levels.It shows that gga-miR-451 inhibits the proliferation and cycle progression of MG-HS-infected DF-1 cells,promotes cell apoptosis and inhibits MG-HS propagation in DF-1 cells.7.RT-q PCR results showed that p MGA1.2 was highly expressed in time-dependent at 24,48,and 72 h post-infection.The primary transcript of gga-miR-451(pri-miR-451)and precursor(pre-miR-451)expression levels were significantly up-regulated with no time-dependent increase in DF-1 cells following MG-HS infection,indicating that the upregulation of gga-miR-451 may happen at the transcriptional level.The results of dual-luciferase reporter showed that there is a major transcription factor binding site within 386-254 bp upstream of the transcription site,which is required for gga-miR-451 transcriptional activity.Gene-regulation and JASPAR were used to analyze the putative binding sequences.The results of dual-luciferase reporter showed that the three Ah R:Arnt binding sites are necessary for the activation of gga-miR-451 promoter by MG-HS infection.The results of dual-luciferase reporter and Ch IP showed that Ahr:Arnt enhanced the transcription activity of gga-miR-451 during MG-HS infection through binding to the gga-miR-451 promoter region.RT-q PCR and WB further verified that the overexpression of Ah R:Arnt significantly up-regulated gga-miR-451 expression and significantly inhibited YWHAZ expression.These data demonstrate that Ah R:Arnt binds to the core promoter of gga-miR-451,induces the expression of mature gga-miR-451,and eventually suppresses YWHAZ expression.